Anti ganglioside GD3 antibodies and uses thereof

ABSTRACT

The present invention is related to complementarity determining region (cdr)-grafted humanized r24 antibodies that bind to the gd3 ganglioside antigen. The humanized antibodies disclosed herein have characteristics that are comparable or superior to the murine r24 antibody, and the humanized antibodies are useful in treating cancer (e.g. Melanoma).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International applicationNo. PCT/US2008/54220, filed Feb. 18, 2008, which claims the benefit ofpriority of U.S. Ser. No. 60/901,875, filed Feb. 16, 2007. The entirecontents and disclosures of the preceding applications are incorporatedby reference into this application.

Throughout this application, various references or publications arecited. Disclosures of these references or publications in theirentireties are hereby incorporated by reference into this application inorder to more fully describe the state of the art to which thisinvention pertains.

FIELD OF THE INVENTION

The present invention relates to the making and using of humanizedantibodies that bind to the ganglioside GD3 antigen.

BACKGROUND OF THE INVENTION

The GD3 ganglioside is a glycosphingolipid that is over expressed onmelanoma cells. Thus, the GD3 ganglioside is an attractive target forimmunotherapy of melanoma. At present, there is no effective therapeuticregimen approved for advanced melanoma which results in a sustainedbenefit. The treatment guidelines that exist in most countries focusprimarily on surgery to remove malignant melanoma and there is little ifany guidance on the selection of chemotherapy or immunotherapy regimens.Even between experts, there is little if any agreement on treatmentoptions.

In Stages I-III of the disease, surgery is generally the first linetreatment, followed by adjuvant (post-resection) treatment usingrecombinant IFN alpha2 products (Schering Plough's Intron A™), orvarious combinations of chemotherapeutic regimens, although there is nodemonstrated sustained survival benefit in the rIFN treatment regimens.For Stage 1V patients, where the disease has metastasised, treatmentsused tend to be chemotherapy or high-dose IFN alpha2, but the prognosisfor such patients is very poor, with a very low (9%) response rate and amedian survival rate of six to nine months. A significant proportion ofStage II+III patients also are at risk of developing metastatic disease.Due to the bleak outlook for Stage IV patients, most are enrolled inclinical trials for developing therapeutics.

Previous work has identified a mouse IgG3 monoclonal antibody, known asR24, that binds to the GD3 ganglioside. This antibody has been shown toexhibit a phenomenon known as homophilic binding, whereby the antibodiesbind to each other. Furthermore, although IgG3 mouse antibodies are notparticularly efficient at fixing human complement, it has been foundthat R24 was active in vitro with respect to complement-dependentcytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)(Chapman et al., J. Immunol. (1990) 145 (3): 891-898).

The murine R24 antibody has been used in the treatment of malignantmelanoma. A considerable number of clinical trials have been conductedwith the murine R24 antibody in patients with late stage malignantmelanoma, either alone or in combination with other agents, and thesehave had some success with regards to tumour regression. However, theantibody elicited some immunogenic responses in a significant proportionof patients owing to the fact that it was murine in nature (Bajorin, D.F. et al., Melanoma Research (1992) 2 (5-6): 355-362). For this reason,dosing was restricted and the treatment regimens were not able to beoptimised.

A chimeric mouse-human version of the R24 antibody has been generated.However, this chimeric antibody exhibited a substantially lower level ofbinding to the GD3-binding site compared to the murine R24 antibody. Inaddition, while the chimeric antibody maintained the characteristics ofhomophilic binding and fixing human complement, the efficiency of ADCCwas slightly reduced. This chimeric antibody is formed from entiremurine antibody heavy chain and light chain variable regions engineeredonto human heavy chain and light chain constant regions as described inChapman et al., Cancer Immunol. Immunother. (1994) 39: 198-204.

There are other known monoclonal mouse antibodies that bind to the GD3ganglioside, such as the KM-641 antibody produced by a hybridoma KM-641.Chimeric versions of these antibodies, e.g., KM-871, are also known (seeU.S. Pat. No. 6,437,098). These chimeric versions are also formed fromentire murine antibody heavy chain and light chain variable regionsengineered onto human heavy chain and light chain constant regions.

Most therapies in development are targeting the earlier, non-metastaticstages of malignant melanoma, owing to the difficulties in obtaining aresponse when the disease has metastasised. There are very fewimmunotherapeutics targeting Stage IV melanoma.

Thus, there is clearly a significant demand for an effective treatment,particularly for advanced malignant melanoma, but also melanomagenerally.

SUMMARY OF THE INVENTION

The present invention provides an antibody comprising a heavy chainvariable region comprising the following amino acid sequence:

(SEQ ID NO: 1 or 3) Q/DVQLVESGGGV/LVQPGR/GSL/RR/KLSCAASGFTFSNFGMHWVRQAPG/EKGLEWVAYISSGGSSINYADTVKGRFTISRDNS/PKNTLY/FLQMN/TSLRA/SEDTAV/IYYCTRGGTGTRSLYYFDYWGQGT/ATV/LT/IV,a fragment of said sequence or a sequence that is homologous thereto;and a light chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 2 or 4) DIQMTQSPSSLSASVGDRVTITCRASQDIGNFLNWYQQKPGKAPKLLI/LYYTSRLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGKT LPYTFGGGTKVEIK,a fragment of said sequence or a sequence that is homologous thereto.

In one embodiment there is provided an antibody comprising a heavy chainvariable region comprising the following amino acid sequence:

(SEQ ID NO: 24 or 25) Q/DVQLVESGGGV/LVQPGR/GSL/RR/KLSCAASGFTFSNFGMHWVRQAPG/EKGLEWVAYISSGGSSINYADTVKGRFTISRDNS/PKNTLY/FLQMN/TSLRA/SEDTAV/IYYCTRGGTGTRSLYYFDYWGQGT/ATV/LT/ IV SS,a fragment of said sequence or a sequence that is homologous thereto;and a light chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 2 or 4) DIQMTQSPSSLSASVGDRVTITCRASQDIGNFLNWYQQKPGKAPKLLI/LYYTSRLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGKTLPY TFGGGTKVEIK,a fragment of said sequence or a sequence that is homologous thereto.

In one embodiment, the antibody of the present invention comprises aheavy chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 1) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NFGMHWVRQA PGKGLEWVAYISSGGSSINY ADTVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRGG TGTRSLYYFDYWGQGTTVTV,and a light chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 2) DIQMTQSPSS LSASVGDRVT ITCRASQDIG NFLNWYQQKP GKAPKLLIYYTSRLQSGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GKTLPYTFGG GTKVEIK.

In another embodiment, the antibody of the present invention comprises aheavy chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 3) DVQLVESGGG LVQPGGSRKL SCAASGFTFS NFGMHWVRQA PEKGLEWVAYISSGGSSINY ADTVKGRFTI SRDNPKNTLF LQMTSLRSED TAIYYCTRGG TGTRSLYYFDYWGQGATLIV;and a light chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 4) DIQMTQSPSS LSASVGDRVT ITCRASQDIG NFLNWYQQKP GKAPKLLLYYTSRLQSGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GKTLPYTFGG GTKVEIK.

The present invention also provides compositions comprising theantibodies disclosed herein. In one embodiment, the antibodies areradio-labeled. In another embodiment, the compositions further comprisea therapeutic or an anti-melanoma drug. Examples of the therapeutic oranti-melanoma drug include, but are not limited to, one or morecytotoxic drugs, interleukins, drugs that activate the immune system orlymphocytes, antibodies that bind to melanoma, antibodies that bind totumor matrix, antibodies designed to inhibit vascular structures, anddrugs that block critical biochemical pathways in melanoma. Accordingly,examples of the therapeutic or anti-melanoma drug include, but are notlimited to, dacarbazine, fotemustine, carmustine, lomustine,temozolomide, amifostine, cisplatin, carboplatin, taxol, taxotere,tamoxifen, vinblastin, vincristine, DHFR inhibitors (e.g. piritreximisethionate), interleukin 2 including aldesleukin, interferon alpha,interferon alpha-2b, other anti-tumor antibodies or kinase inhibitorssuch as tyrosine kinase inhibitors or serine/threonin kinase inhibitors.

The present invention also provides isolated nucleic acid sequencesencoding the antibodies disclosed herein, as well as vectors comprisingthe nucleic acid sequences, and host cells comprising the vectors. Inone embodiment, the host cells are avian cells, plant cells, algae,fungal cells, or animal cells.

The present invention also provides methods of using the antibodiesdisclosed herein to treat malignant diseases such as malignant melanoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates binding of humanized R24 antibodies to MeWo humanmelanoma cell line over a range of antibody concentrations 100ug/ml-0.0125 ug/ml, each concentration was performed in triplicate andthe results are presented as mean % MLF+/−standard error.

FIG. 2 illustrates the binding of the LD49 antibody, the murine R24antibody and a previously published chimeric antibody to the SK-MEL28cells.

FIG. 3 illustrates Biosensor analysis of the interaction betweenhumanized antibodies LD41-LD52 and immobilized GD3. The ‘Off’ rates werenormalized to give identical ‘injection end point’ RU values.

FIG. 4 illustrates binding of LD47 and LD49 humanized R24 antibodies tohuman ganglioside GD3.

FIG. 5 illustrates lysis of MeWo cells by complement. The experiment wasperformed in triplicate and the results are presented as mean %cytotoxicity+/−standard error.

FIG. 6 illustrates homophilic binding of humanized R24 antibodies tomouse R24.

FIG. 7 illustrates the effect of antibody concentration and E:T ratio onLD49's ability to mediate ADCC.

FIG. 8 illustrates ADCC over a range of E:T ratios. The experiment wasperformed in triplicate and the results are presented as mean %cytotoxicity+/standard error.

FIG. 9 illustrates ADCC over a range of antibody concentrations. Theexperiment was performed in triplicate and the results are presented asmean % cytotoxicity+/−standard error.

FIG. 10 illustrates repeated ADCC experiments with (shaded) and without(white) PBMC for humanized LD47 and LD49 R24 antibodies. The experimentwas performed in triplicate and the results are presented as mean %cytotoxicity+/−standard error.

DETAILED DESCRIPTION OF THE INVENTION

Humanization (CDR-grafting) is now a well-established technique forreducing the immunogenicity of monoclonal antibodies (mAbs) fromxenogeneic sources (commonly rodent), and for improving their activationof the human immune system. The technique involves grafting rodentantibody complementarity-determining regions (CDRs) into human antibodyvariable domain frameworks. However, such a technique does not alwaysreconstitute the binding affinity and specificity of the original mAb.The design of the humanized antibody is important for reproduction ofthe function of the original donor antibody, and includes variouschoices: the extents of the CDRs, the human frameworks to use and thesubstitution of residues from the rodent mAb into the human frameworkregions (back mutations). As such the procedure is complicated and it isdifficult to predict success.

It has now been surprisingly found that R24 complementarity determiningregion (CDR)-grafted humanized antibodies have characteristics that arecomparable or superior to the murine R24 antibody including exhibitingsimilar binding affinity to the GD3 ganglioside antigen.

According to one aspect of the present invention, there is provided anantibody comprising a heavy chain variable region comprising thefollowing amino acid sequence:

(SEQ ID NO: 1 or 3) Q/DVQLVESGGGV/LVQPGR/GSL/RR/KLSCAASGFTFSNFGMHWVRQAPG/EKGLEWVAYISSGGSSINYADTVKGRFTISRDNS/PKNTLY/FLQMN/TSLRA/SEDTAV/IYYCTRGGTGTRSLYYFDYWGQGT/ATV/ LT/IV,

a fragment of said sequence or a sequence that is homologous thereto;and a light chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 2 or 4) DIQMTQSPSSLSASVGDRVTITCRASQDIGNFLNWYQQKPGKAPKLLI/LYYTSRLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGKTLPYT FGGGTKVEIK,

a fragment of said sequence or a sequence that is homologous thereto.

An antibody of the present invention can have a heavy chain variableregion with the amino acids glutamine or aspartic acid at position 1,valine or leucine at position 11, arginine or glycine at position 16,leucine or arginine at position 18, arginine or lysine at position 19,glycine or glutamic acid at position 42, serine or proline at position74, tyrosine or phenylalanine at position 79, asparagine or threonine atposition 82, alanine or serine at position 84, valine or isoleucine atposition 89, threonine or alanine at position 107, valine or leucine atposition 109 and threonine or isoleucine at position 110 and a lightchain variable region with the amino acids isoleucine or leucine atposition 48. The positions of the amino acids are described withreference to Kabat numbering as described in Kabat, E. A. et al.,Sequences of Proteins of Immunological Interest, NIH publication (1991)Fifth edition, No. 91-3242.

In one embodiment, an antibody of the present invention comprises aheavy chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 1) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NFGMHWVRQA PGKGLEWVAYISSGGSSINY ADTVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRGG TGTRSLYYFDYWGQGTTVTV;

and a light chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 2) DIQMTQSPSS LSASVGDRVT ITCRASQDIG NFLNWYQQKP GKAPKLLIYYTSRLQSGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GKTLPYTFGG GTKVEIK

In another embodiment, an antibody of the present invention comprises aheavy chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 3) DVQLVESGGG LVQPGGSRKL SCAASGFTFS NFGMHWVRQA PEKGLEWVAYISSGGSSINY ADTVKGRFTI SRDNPKNTLF LQMTSLRSED TAIYYCTRGG TGTRSLYYFDYWGQGATLIV;

and a light chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 4) DIQMTQSPSS LSASVGDRVT ITCRASQDIG NFLNWYQQKP GKAPKLLLYYTSRLQSGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GKTLPYTFGG GTKVEIK

In one embodiment, an antibody of the present invention comprises aheavy chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 1) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NFGMHWVRQA PGKGLEWVAYISSGGSSINY ADTVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRGG TGTRSLYYFDYWGQGTTVTV;

and a light chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 4) DIQMTQSPSS LSASVGDRVT ITCRASQDIG NFLNWYQQKP GKAPKLLLYYTSRLQSGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GKTLPYTFGG GTKVEIK

In another embodiment, an antibody of the present invention comprises aheavy chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 3) DVQLVESGGG LVQPGGSRKL SCAASGFTFS NFGMHWVRQA PEKGLEWVAYISSGGSSINY ADTVKGRFTI SRDNPKNTLF LQMTSLRSED TAIYYCTRGG TGTRSLYYFDYWGQGATLIV;

and a light chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 2) DIQMTQSPSS LSASVGDRVT ITCRASQDIG NFLNWYQQKP GKAPKLLIYYTSRLQSGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GKTLPYTFGG GTKVEIK

An antibody is a protein made up of four polypeptide chains: twoidentical heavy (also known as gamma for IgG isotype antibodies) chainsand two identical light (also known as kappa or lambda) chains connectedby disulfide bonds. Each heavy and light chain has a constant region anda variable region. The CDRs are found within the variable regions. TheCDRs will be in contact with an antigen, when an antibody binds to anantigen.

The antibodies of the present invention recognize and bind to the GD3ganglioside antigen. Antibodies of the present invention may comprise atleast one human heavy chain variable region or at least one human lightchain variable region in which the human CDR sequence is replaced by R24murine antibody CDR sequence. In addition, the antibody of the inventioncan contain 1, 2, 3, 4 or 5 mutations back to the murine R24 antibodysequence in the non-CDR human heavy chain or human light chain variableregions

The antibodies of the invention as defined above comprisecomplementarity determining regions (CDRs) with the following amino acidsequences in the heavy chain variable region:

NFGMH, (SEQ ID NO: 5) YISSGGSSINYADTV (SEQ ID NO: 6) and GGTGTRSLYYFDY;(SEQ ID NO: 7)and the following amino acid sequences in the light chain variableregion:

RASQDIGNFLN, (SEQ ID NO: 8) YTSRLQS (SEQ ID NO: 9) and QQGKTLPYT. (SEQID NO: 10)

A fragment of an antibody described above comprises at least one of theCDRs described above and retains the ability to bind to the GD3ganglioside antigen. Specific examples of binding fragments are i) theFab fragment that consists of the VL, VH, CL and CH1 domains; ii) the Fdfragment consisting of the VH and CH1 domains; iii) the Fv fragmentconsisting of VL and VH domains; iv) the dAB fragment (Ward, E. S. etal., Nature (1989) 341:544-546), which consists of a VH domain; v)isolated CDR regions; vi) F(ab′)2 fragments, a bivalent fragmentcomprising two linked Fab fragments, vii) single chain Fv molecules(scFv), wherein a VH domain and a VL domain are linked by a peptidelinker which allows the two regions to associate to form an antigenbinding site (Bird et al., Science (1988) 242:423-426 and Huston et al.,PNAS USA (1988) 85:5879-5883); viii) bispecific single chain Fv dimers(PCT/US92/09965) and (ix) “diabodies”, multivalent or multispecificfragments constructed by gene fusion (W094/13804 and Hollinger et al.,(1993) Proc. Natl. Acad. Sci. USA, 90:6444-6448).

A fragment of an antibody of the present invention is a stretch of aminoacid residues of at least 5 to 7 contiguous amino acids, often at least7 to 9 contiguous amino acids, typically at least about 9 to 13contiguous amino acids and preferably at least about 20 to 30 or morecontiguous amino acids and most preferably at least about 30 to 40 ormore consecutive amino acids.

One aspect of the present invention extends to a heavy chain variableregion with an amino acid sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, 99.5% or 99.9% homologous to the sequence of theheavy chain variable region described above. Preferably the heavy chainvariable region is 90% homologous to the sequence of the heavy chainvariable region described above.

One aspect of the present invention extends to a light chain variableregion with an amino acid sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, 99.5% or 99.9% homologous to the sequence of thelight chain variable region described above. Preferably the light chainvariable region is 90% homologous to the sequence of the light chainvariable region described above.

The antibodies of the present invention further comprise a heavy chainconstant region and a light chain constant region of a human antibody.Human heavy chain constant regions may be from one of five classes (IgM,IgG, IgA, IgE or IgD) or their sub-classes (IgG1, IgG2, IgG3, IgG4, IgA1or IgA2). Light chain constant regions may be from the kappa or lambdaclasses. The antibodies of the invention can be human IgG1/kappaantibodies, for example. Heavy chain and light chain constant regionsfrom non-human antibodies may also be used with the variable domains ofthe invention, if those constant regions have been deimmunized for usein man.

In one embodiment, the heavy chain constant region of the antibodies maycomprise the following amino acid sequence:

(SEQ ID NO: 11) DIPSTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGALTSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKTHTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVEVHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQPREPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

In one embodiment, the light chain constant region of the antibodies maycomprise the following amino acid sequence:

(SEQ ID NO: 12) DIR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQWKVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

A further embodiment of the invention is an antibody which comprises aheavy chain region comprising the following amino acid sequence:

(SEQ ID NO: 13 or 15) Q/DVQLVESGGG V/LVQPGR/GSL/RR/KL SCAASGFTFSNFGMHWVRQA PG/EKGLEWVAY ISSGGSSINY ADTVKGRFTI SRDNS/PKNTLY/FLQMN/TSLRA/SED TAV/IYYCTRGG TGTRSLYYFD YWGQGT/ATV/LT/IV SSDIPSTKGPSVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLSSVVTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKT HTCPPCPAPE LLGGPSVFLFPPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVVSVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVFSCSVMHEALH NHYTQKSLSL SPGK

and a light chain region comprising the following amino acid sequence:

(SEQ ID NO: 14 or 16) DIQMTQSPSS LSASVGDRVT ITCRASQDIG NFLNWYQQKPGKAPKLLI/LYY TSRLQSGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GKTLPYTFGGGTKVEIKDIR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

This embodiment of the invention extends to a heavy chain region with anamino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, 99.5% or 99.9% homologous to the sequence of the heavy chain regiondescribed above. Preferably the heavy chain region is 97% homologous tothe sequence of the heavy chain region described above.

This embodiment of the invention also extends to a light chain regionwith an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, 99.5% or 99.9% homologous to the sequence of the lightchain region described above. Preferably the light chain region is 97%homologous to the sequence of the light chain region described above.

In another embodiment, the present invention provides an antibodydesignated LD49, which comprises a heavy chain region comprising thefollowing amino acid sequence:

(SEQ ID NO: 13) DVQLVESGGG LVQPGGSRKL SCAASGFTFS NFGMHWVRQA PEKGLEWVAYISSGGSSINY ADTVKGRFTI SRDNPKNTLF LQMTSLRSED TAIYYCTRGG TGTRSLYYFDYWGQGATLIV SSDIPSTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGALTSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKTHTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVEVHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQPREPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK;

and a light chain region comprising the following amino acid sequence:

(SEQ ID NO: 14) DIQMTQSPSS LSASVGDRVT ITCRASQDIG NFLNWYQQKP GKAPKLLLYYTSRLQSGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GKTLPYTFGG GTKVEIKDIRTVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDSKDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

Still yet another embodiment of the invention is an antibody designatedLD47, which comprises a heavy chain region comprising the followingamino acid sequence:

(SEQ ID NO: 15) QVQLVESGGG VVQPGRSLRL SCAASGFTFS NFGMHWVRQA PGKGLEWVAYISSGGSSINY ADTVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCTRGG TGTRSLYYFDYWGQGTTVTV SSDIPSTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGALTSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKTHTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVEVHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQPREPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK;

and a light chain region comprising the following amino acid sequence:

(SEQ ID NO: 16) DIQMTQSPSS LSASVGDRVT ITCRASQDIG NFLNWYQQKP GKAPKLLIYYTSRLQSGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GKTLPYTFGG GTKVEIKDIRTVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDSKDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

A further embodiment of the invention is an antibody comprising avariant of the heavy chain variable region and/or heavy chain constantregion as described above and a variant of the light chain variableregion and/or light chain constant region as described above.

A variant of the heavy chain and light chain variable or constantregions of an antibody of the present invention is understood to meanthe sequences of the heavy chain and light chain variable or constantregions with one or more deletions, insertions, or substitutions.Particular embodiments include, but are not limited to, heavy chain andlight chain variable and constant regions described above that comprisefrom one to ten deletions, insertions or substitutions of amino acidresidues, when compared to the heavy chain and light chain variable andconstant regions described above. In one embodiment, a variant has anamino acid sequence that is at least 85% identical to the heavy chainand light chain variable regions described above and in anotherembodiment the variant has an amino acid sequence that is at least 90%identical to the heavy chain and light chain variable regions describedabove. The percent identity may be determined, for example, by comparingsequence information using the GAP computer program, version 6.0described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman(Adv. Appl. Math 2:482, 1981). The preferred default parameters for theGAP program include: (1) a unary comparison matrix (containing a valueof 1 for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps. Other programs used by one skilled in the art of sequencecomparison may also be used.

A given amino acid may be replaced by any residue that does notadversely affect antibody function. The replacement residue can havesimilar physiochemical characteristics to the original residue i.e. aconservative substitution, although this is not essential. Examples ofsuch conservative substitutions include substitution of one aliphaticresidue for another, such as Ile, Val, Leu, or Ala for one another;substitutions of one polar residue for another, such as between Lys andArg, Glu and Asp, or Gln and Asn; or substitutions of one aromaticresidue for another, such as Phe, Trp, or Tyr for one another. Otherconservative substitutions, e.g., involving substitutions of entireregions having similar hydrophobicity characteristics, are well known.

The percent identity of two amino acid sequences or of two nucleic acidsequences may be determined by aligning the sequences for optimalcomparison purposes (e.g. gaps can be introduced in the first sequencefor best alignment with the sequence) and comparing the amino acidresidues or nucleotides at corresponding positions. The “best alignment”is an alignment of two sequences which results in the highest percentidentity. The percent identity is determined by the number of identicalamino acid residues or nucleotides in the sequences being compared(i.e., % identity=number of identical positions/total number ofpositions×100).

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm known to those skilled inthe art. The NBLAST and XBLAST programs are examples of computerprograms which perform such algorithms. BLAST nucleotide searches can beperformed with the NBLAST program to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program to obtain amino acidsequences homologous to protein molecules of the invention.

To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized. Alternatively, PSI-Blast can be used to perform an iteratedsearch which detects distant relationships between molecules (Idem.).When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the defaultparameters of the respective programs (e.g., XBLAST and NBLAST) can beused. Further suitable computer based algorithm programs can be utilizedand will be known to the person skilled in the art.

The antibody described above or variants thereof can be modifiedpost-translationally. Post-translational modification means theenzymatic processing of a polypeptide chain such as an antibodyfollowing its translation from its mRNA transcript. Thepost-translational modification may include the following activities:

(i) glycosylation which relates to the addition of carbohydrate moietiesto the antibody molecule,

(ii) phosphorylation which relates to the addition of a phosphatemolecular group to the antibody molecule,

(iii) sulfation which is the addition of sulfate molecular groups to theantibody,

(iv) acetylation which is the addition of an acetyl molecular group tothe antibody,

(v) ribosylation which is the addition of a ribose molecular group tothe antibody, and

(vi) cleavage which is the removal of a portion of the polypeptide chainand/or fragmentation of a single polypeptide into two or more fragmentsin order to produce a functional antibody in the correct environment.

Antibodies against tumor antigens can also be coupled to radioactiveisotopes for therapeutic purposes, radioimmunotherapy. When infused intoa patient, or delivered directly into a solid tumor or around a solidtumor, these radiation-carrying antibodies circulate in the body untilthey locate and bind to the surface of specific cells, and then delivertheir cytotoxic radiation directly to the cancerous cells.Radioimmunotherapy has been already successfully employed for thetreatment of several cancers, for example with the use of ZEVALIN, anYttrium-90 labeled monoclonal antibody.

Currently, beta radiation emitting radionuclides are used almostexclusively in radioimmunotherapy. This is due to their relatively longpath length in biological tissue (in the mm range), which is sufficientto irradiate cancer cells that do not have bound radiolabeled antibody.Beta-emitters provide a relatively uniform radiation dose to the tumour.However, the shorter-range alpha radiation emitters (50-100 mm) havebeen shown to be more efficient than beta-emitters at inducing lethallesions in single cells. Therefore alpha emitters can also provide afavorable therapeutic index, for example in single tumour cells in thecirculation, micrometastases and in certain cases, minimal residualdisease. Therefore both alpha and beta emitter atoms can be coupled tothe antibodies of the present invention to create a radioimmunotherapy.

In one embodiment of the present invention the antibodies of the presentinvention are labeled with any one of the following isotopes:Phosphorus-32 (beta), Scandium-47, Strontium-89 (beta), Yttrium-90(beta), Rhodium-105 (beta and gamma), Tin-117m (gamma), Iodine-131 (betaand gamma), Samarium-153 (beta), Dysprosium-166 (beta and gamma),Erbium-169 (beta), Ytterbium-175 (beta), Lutetium-177, Rhenium-188(beta), Osmium-194, Bismuth-212 (alpha), Bismuth-213 (alpha),Actinium-225 (alpha).

In another embodiment of the present invention the radiolabeledantibodies of the present invention are used for therapeutic purposesfor the treatment of cancer.

There are many descriptions in the art on how to radiolabel antibodiesfor diagnostic or therapeutic purposes. Therefore it is considered to bewell known in the art how to radiolabel an antibody and one would not besubjected to undue experimentation to create the radiolabeled antibodiesof the present invention. For example, Fritzberg et al (Approaches toRadiolabeling of Antibodies for Diagnosis and Therapy of CancerPharmaceutical Research, Volume 5, Number 6/June, Pages 325-334, 1988)already describes methods for such labeling. U.S. Pat. No. 7,229,620specifically describes methods to radiolabel an antibody withYttrium-90. In “Direct radiolabeling of monoclonal antibodies withRhenium-188 for radioimmunotherapy of solid tumors a review ofradiolabeling characteristics, quality control and in vitro stabilitystudies” (Applied Radiation and Isotopes Volume 54, Issue 3, March 2001,Pages 399-406), it is described a method to radiolabel an antibody withRhenium-188.

Methods for radiolabeling of antibodies can be grouped as follows: 1)the direct labeling in which the inner di-sulfide bonds (—S—S—) of thehinge-region of the antibodies are being partially reduced to sulfhydrilgroups (—SH). To do this, various chemical compounds having reducingproperties are used, such as for example, the derivatives of ascorbicacid (Hnatowich D. et al. J. Nucl. Med. 1994; 35: 127-134), and/orsubstances with sulfhydryl groups or stannic-II-compounds or complexes(Mather S. et al. J. Nucl. Med. 1990; 31: 692-697, Rhodes B. J. Nucl.Med. 1986:27:685-693, and Thakur M. et al. Nucl. Med. Biol. 1991; 18:227-233,) and 2) the indirect labeling methods in which mostlybi-functional complex forming agents are used, such as diamine-dithiol(Baidoo K. et al. Cancer Res. 1990:50:799-803), or a bi-functional esterof NHS-BAT (Eisenhut M. et al. J Nucl Med 1991; 37: 362-370), ordiamid-dimercaptid (Kasina S. et al. J. Nucl. Med. 1991; 32: 1445-1451)and/or DTPA (Najafi A. et al. Int. J. Appl. Radiat. Isot. 1984; 5:554-557), or a novel complex forming agent, which is based upon aN2S4-composition (Najafi A. et al. Nucl. Med. Biol. 1991; 18: 179-185,Qu T. et al. Radiochim. Acta 1993; 63: 209-212) for conjugating theradionuclide onto the antibodies. Other methods for indirect conjugationof the radionuclide to an antibody are based on the conjugation ofthiol-groups to amino acids (e.g. lysine) in the protein molecule with2-iminothiolan (Joiris E. et al. Nucl. Med. Biol. 1991, 18: 353-356) orwith the groups of 1-imino-4-mercaptobutyl compounds (Goedemans W. inNicolin M. et al. (eds.) Verona 1990; 595-603). Suitable complex formingagents for the complex formation, especially with Yttrium (preferablyY-90) are, for example, DOTA (Denora et al. Anticancer Research 1997,17, 1735-1744) or 12 N4-maleimid (tetra-azocyclododecantextra-aceticacid) (Turner et al. Br. J. Cancer, 1994, 70: 35-41, and King et al.Cancer Research, 1994, 54: 6176-6185). Particularly suited for complexformation of Rhenium (preferably Re-186 or Re-188), is for example, theMAG-3 complex forming agent (van Gog et al. J. Nucl. Med. 1996, 37, (2),352-362). A chelator such as CHX-DTPA is disclosed in U.S. Pat. No.5,641,471 for preparing and antibody with attached Bismuth-213. U.S.Pat. No. 5,246,691 discloses chelating agents to create antibodies withattached Actinium-225

Many isotopes like Phosphorus-32, Strontium-89, Yttrium-90,Samarium-153, Erbium-169, Ytterbium-175, Rhenium-188 have been stablycoupled to antibodies for therapeutic purposes by means of bi-functionalcomplex forming agents (while iodine has been known for many years tocouple to antibodies, it is not particularly useful because of itsunfavorable radiation properties).

Radiolabeled antibodies are important clinical reagents for both tumorimaging and therapy since they provide a direct mode of destruction bydirecting destructive radioactive energy to the cancer site via a homingantibody. For example, three radiolabeled antibodies two murine, Y-90ibritumomab tiuxetan (Zevalin; Biogen Idec) and I-131 tositumomab(Bexxar; Corixa/GSK), and one chimeric, I-131 ch-TNT (Shanghai MedipharmBiotech) have been approved for non-Hodgkin's lymphoma or lung cancer.

It is an object of the present invention to use the radiolabeledantibody of the present invention as a therapy for cancer.

In another aspect, the present invention further extends to an antibodyin combination with at least one different therapeutic or anti-melanomadrug. In one embodiment the therapeutic or anti-melanoma drug is acytotoxic chemotherapy drug or combination of drugs such as dacarbazine,temozolomide, fotemustine, carmustine, lomustine, temozolomide,cisplatin, carboplatin, TAXOL, TAXOTERE, tamoxifen, vinblastin,vincristine, and DHFR inhibitors (e.g. piritrexim isethionate). Anotherembodiment is a drug to activate the immune system such as interleukin 2(such as ALDESLEUKIN), interferon alpha and interferon alpha-2b, othercytokines/chemokines, or agents to activate lymphocytes such asIPILIMUMAB, TICILIMUMAB or other drugs designed to activate lymphocytes.In another embodiment, the drug is another monoclonal antibody thatbinds to melanoma, such as anti-GD2 antibodies or anti-HMW-MAAantibodies, antibodies that bind to tumor matrix, or antibodies designedto inhibit vascular structures such as BEVACICUMAB. Another embodimentis to add drugs that block critical biochemical pathways in melanoma,such as the MAPK pathway or the AKT/PI3K pathways. Examples of thesedrugs include, but are not limited to: SORAFENIB, AZD6244, PLX4032,XL281, PD0325901, SUNITINIB, and GLEEVEC.

The present invention also provides a method of treatment and/orprophylaxis of malignant melanoma, the method comprising the step ofadministering an antibody described above. The malignant melanoma can beadvanced stage malignant melanoma, which is stage IV of the disease.Malignant melanoma is the most aggressive form of skin cancer. It is amalignant tumor of melanocytes, the cells which make melanin, thepigment that gives skin its colour and protects it from ultravioletradiation. Malignant melanoma most commonly occurs in the skin but mayalso occur at any site to which melanocytes have migrated. Lessfrequently, the cancer may occur in the melanocytes of the retinalpigment epithelial cells (uveal melanoma). There are four main ‘stages’of disease progression, Stages I to IV, with treatment becoming moreproblematic the more advanced the disease becomes.

The method of treatment of malignant melanoma with an antibody of theinvention further comprises the step of treating a subject with othertherapy used to treat melanoma such as radiation therapy or surgery. Theother therapy such as radiation therapy can be administeredsimultaneous, separate or sequential to administration of an antibody ofthe invention to a subject.

The method of treatment can be in relation to a subject that haspreviously undergone surgery to treat the malignant melanoma.Alternatively, the method of treatment can be as a first line oftreatment of malignant melanoma i.e. where the subject has notpreviously undergone surgery for treating the malignant melanoma.

This aspect of the invention includes treatment with an antibodydescribed above in combination with at least one different therapeuticor anti-melanoma drug. The therapeutic or anti-melanoma drug may or maynot be approved for treatment of malignant melanoma. In one embodimentthe therapeutic or anti-melanoma drugs are those listed above. Theantibody and the therapeutic or anti-melanoma drug can be administeredsimultaneously, separately or sequentially.

This aspect of the invention further extends to an antibody describedabove for treating malignant melanoma. This aspect also extends to theuse of an antibody described above in the manufacture of a medicamentfor treating malignant melanoma.

This aspect of the invention also extends to a pharmaceuticalcomposition comprising an antibody as described above. The compositionor pharmaceutical composition may optionally further comprise apharmaceutically-acceptable excipient, diluent or carrier.

Treatment/Therapy

The term “treatment” is used herein to refer to any regimen that canbenefit a human or non-human animal. The treatment may be in respect ofan existing condition or may be prophylactic (preventative treatment).Treatment may include curative, alleviation or prophylactic effects.

More specifically, reference herein to “therapeutic” and “prophylactic”treatment is to be considered in its broadest context. The term“therapeutic” does not necessarily imply that a subject is treated untiltotal recovery. Similarly, “prophylactic” does not necessarily mean thatthe subject will not eventually contract a disease condition.

Accordingly, therapeutic and prophylactic treatment includesamelioration of the symptoms of a particular condition or preventing orotherwise reducing the risk of developing a particular condition. Theterm “prophylactic” may be considered as reducing the severity or theonset of a particular condition. “Therapeutic” may also reduce theseverity of an existing condition.

Administration

The antibody of the invention or variants may be administered to apatient in need of treatment via any suitable route. Route ofadministration may include; parenterally (including subcutaneous,intramuscular, intravenous, by means of, for example, a drip patch),some further suitable routes of administration include (but are notlimited to) oral (including buccal and sublingual), rectal, nasal,topical, infusion, vaginal, intradermal, intraperitoneally,intracranially, intrathecal, intra-ocular or eyedrops, and epiduraladministration or administration via oral or nasal inhalation, by meansof, for example a nebulizer or inhaler, or by an implant, or by isolatedlimb perfusion.

For administration via the oral or nasal inhalation routes the antibodymay be delivered using a mechanical form including, but not restrictedto, an inhaler or nebulizer device.

Further, where the oral or nasal inhalation routes are used,administration by a SPAG (small particulate aerosol generator) may beused.

For intravenous injection, the antibody composition ingredient will bein the form of a parenterally acceptable aqueous solution which ispyrogen-free and has suitable pH, isotonicity and stability. Those ofrelevant skill in the art are well able to prepare suitable solutionsusing, for example, isotonic vehicles such as sodium chloride injection,Ringer's injection, Lactated Ringer's injection. Preservatives,stabilizers, buffers, antioxidants and/or other additives may beincluded, as required.

Dose

The composition/antibody is preferably administered to an individual ina “therapeutically effective amount”, this being sufficient to showbenefit to the individual.

The actual amount administered, and rate and time-course ofadministration, will depend on the nature and severity of what is beingtreated. Prescription of treatment, e.g. decisions on dosage etc, isultimately within the responsibility and at the discretion of generalpractitioners and other medical doctors, and typically takes account ofthe disorder to be treated, the condition of the individual patient, thesite of delivery, the method of administration and other factors knownto practitioners.

The optimal dose can be determined by physicians based on a number ofparameters including, for example, age, sex, weight, severity of thecondition being treated, the active ingredient being administered andthe route of administration. Typical per patient doses of the antibodyof the invention are between 3 mg and 1000 mg per metre squared perweek.

In another aspect, the present invention relates to a method ofproducing the antibody described above comprising; i) the step oftransfecting a host with a vector comprising a nucleic acid (NA)sequence that encodes the amino acid sequence of the antibody describedabove, ii) isolating the expressed antibody and iii) purifying theantibody. The nucleic acid can be DNA or RNA.

This aspect of the invention extends to the NA sequence encoding anantibody described above. This aspect of the invention also extends to avector comprising the NA sequence encoding an antibody described above.This aspect of the invention further extends to a host cell transformedwith the vector comprising the NA sequence encoding an antibodydescribed above. This aspect of the invention further extends to a hostcell that is an avian cell, such as a chicken, a plant cell or an animalcell

Expression

This aspect of the invention further provides recombinant cloning andexpression vectors containing NA encoding an antibody described above,as well as host cells containing the recombinant vectors. Expressionvectors comprising NA may be used to prepare an antibody, variant orfragment of the invention encoded by the NA. A method for producingantibodies, variants or fragments comprises culturing host cellstransformed with a recombinant expression vector encoding theantibodies, variants or fragments described above, under conditions thatpromote expression of the antibodies, variants or fragments, thenrecovering the expressed antibodies, variants or fragments from theculture.

Any suitable expression system may be employed. The vectors include a NAencoding an antibody, variant or fragment of the invention, operablylinked to suitable transcriptional or translational regulatorynucleotide sequences, such as those derived from a mammalian, avian,microbial, viral, bacterial, or insect gene. Nucleotide sequences areoperably linked when the regulatory sequence functionally relates to theNA sequence. Thus, a promoter nucleotide sequence is operably linked toa DNA sequence if the promoter nucleotide sequence controls thetranscription of the DNA sequence. An origin of replication that confersthe ability to replicate in the desired host cells, and a selection geneby which transformants are identified, are generally incorporated intothe expression vector.

In addition, a sequence encoding an appropriate signal peptide (nativeor heterologous) can be incorporated into expression vectors. A NAsequence for a signal peptide (secretory leader) may be fused in frameto the nucleic acid sequence of the invention so that the NA isinitially transcribed, and the mRNA translated, into a fusion proteincomprising the signal peptide. A signal peptide that is functional inthe intended host cells promotes extracellular secretion of theantibodies, variants or fragments. The signal peptide is cleaved fromthe antibodies, variants or fragments during translation, but allowssecretion of antibodies, variants or fragments from the cell.

Suitable host cells for expression of antibodies include highereukaryotic cells, algae, and yeast. The higher eukaryotic cells can beavian cells and in particular chicken cells. This is described inW02004/047531. In addition, the higher eukaryotic cells can be plantcells or animal cells. Mammalian cells, and in particular CHO cells areparticularly preferred for use as host cells. Appropriate cloning andexpression vectors for use with mammalian, prokaryotic, yeast, fungaland insect cellular hosts are described, for example, in Pouwels et atCloning Vectors: A Laboratory Manual, Elsevier, N.Y., (1986) (ISBN0444904018). Established cell lines of mammalian origin also may beemployed. Examples of suitable mammalian host cell lines include theCOS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinesehamster ovary (CHO) cells, murine NSO cells, HeLa cells, and BHK (ATCCCRL 10) cell lines, human Per.C6 cells, mouse SP210 cells and theCV1IEBNA cell line derived from the African green monkey kidney cellline CV1 (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821,1991).

Established methods for introducing DNA into mammalian cells have beendescribed (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp.15-69). Additional protocols using commercially available reagents, suchas Lipofectamine or Lipofectamine 2000 lipid reagents (Gibco/BRL) orLipofectamine-Plus lipid reagent, can be used to transfect cells(Felgner et aI., Proc. Natl. Acad. Sci. USA 84:74137417, 1987). Inaddition, electroporation can be used to transfect mammalian cells usingconventional procedures, such as those in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring HarborLaboratory Press, 1989). Selection of stable transformants can beperformed using methods known in the art, such as, for example,resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology185:487-511, 1990, describes several selection schemes, such asdihydrofolate reductase (DHFR) to confer resistance to methotrexate.Other examples of selectable markers that can be incorporated into anexpression vector include cDNAs conferring resistance to antibiotics,such as G418 (Geneticin) and hygromycin B. Cells harboring the vectorcan be selected on the basis of resistance to these compounds.

Transcriptional and translational control sequences for mammalian hostcell expression vectors can be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.NA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication.

Additional control sequences shown to improve expression of heterologousgenes from mammalian expression vectors include such elements as theexpression augmenting sequence element (EASE) derived from CHO cells(Monis et al., Animal Cell Technology, 1997, pp. 529-534 and PCTApplication WO 97/25420).

Prokaryotic expression systems can be used for expression of antibodies,variants and fragments of the invention. Prokaryotic expression systemsare particularly useful for the expression of fragments of antibodies ofthe invention. Prokaryotes include gram-negative or gram-positiveorganisms. Suitable prokaryotic host cells for transformation include,for example, E. coli, Bacillus subtilis, Salmonella typhimurium. In aprokaryotic host cell, such as E. coli, antibodies, variants orfragments may include an N-terminal methionine residue to facilitateexpression of the recombinant in the prokaryotic host cell. TheN-terminal methionine may be cleaved from the expressed recombinantantibodies, variants or fragments.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 which contains genes for ampicillin and tetracyclineresistance and thus provides simple means for identifying transformedcells. An appropriate promoter and a DNA sequence are inserted into thepBR322 vector.

The DNA which encodes the antibodies, variants or fragments of theinvention may be cloned in-frame into the multiple cloning site of anordinary bacterial expression vector. Typically the vector would containan inducible promoter upstream of the cloning site, such that additionof an inducer leads to high-level production of the recombinantantibodies, variants or fragments when desired.

For expression of the recombinant antibodies, variants or fragments, thebacterial cells are propagated in growth medium until reaching apre-determined optical density. Expression of the recombinant protein isthen induced which activates expression of proteins from plasmidscontaining a lac operator/promoter. After induction (typically for 1-4hours), the cells are harvested.

For recovery of the expressed antibodies, variants or fragments, thepelleted cells may be resuspended in ten volumes of 50 mM Tris-HCl (pH8)/1 M NaCl and then passed two or three times through a French press.Most highly expressed recombinant antibodies, variants or fragments forminsoluble aggregates known as inclusion bodies. Inclusion bodies can bepurified away from the soluble proteins by pelleting in a centrifuge.The inclusion body pellet is washed and then dissolved in Tris-HCl. Anymaterial that cannot be dissolved is removed by centrifugation.

The protein of interest (i.e. the antibodies, variants or fragments)will, in most cases, be the most abundant protein in the resultingclarified supernatant. This protein may be “refolded” into the activeconformation. After refolding, purification can be carried out by avariety of chromatographic methods.

The antibodies, variants or fragments of the invention may be expressedin yeast host cells, preferably from the Saccharomyces genus (e.g., S.cerevisiae). Other genera of yeast, such as Pichia (pichia pastoris) orKluyveromyces, may also be employed. Yeast vectors will often contain anorigin of replication sequence from a 2 [mu] yeast plasmid, anautonomously replicating sequence (ARS), a promoter region, sequencesfor polyadenylation; sequences for transcription termination, and aselectable marker gene. Suitable promoter sequences for yeast vectorsinclude, among others, promoters for metallothionine, 3-phosphoglyceratekinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or otherglycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; andHolland et aI., Biochem. 17:4900, 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phospho-glucose isomerase, and glucokinase.

Leader sequences suitable for facilitating secretion of recombinantantibodies, variants or fragments from yeast hosts are known to those ofskill in the art. A leader sequence may be modified near its 3′ end tocontain one or more restriction sites. This will facilitate fusion ofthe leader sequence to the structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978.

Isolation and Purification

The invention also includes methods of isolating and purifying theantibody and fragments thereof. An isolated and purified antibodyaccording to the invention can be produced by recombinant expressionsystems as described. Antibodies can be substantially purified, asindicated by a single protein band upon analysis by SDS-polyacrylamidegel electrophoresis (SDS-PAGE).

With respect to any type of host cell, as is known to the skilledartisan, procedures for purifying a recombinant antibody, variant orfragment will vary according to such factors as the type of host cellsemployed and whether or not the recombinant antibody, variant orfragment is secreted into the culture medium.

In general, the recombinant antibodies, variants or fragments can beisolated from the host cells if not secreted, or from the medium orsupernatant if soluble and secreted, followed by one or moreconcentration, salting-out, ion exchange, hydrophobic interaction,affinity purification or size exclusion chromatography steps.

As to specific ways to accomplish these steps, the culture medium firstcan be concentrated using a commercially available protein concentrationfilter, for example, a Millipore Pellicon ultrafiltration unit.Following the concentration step, the concentrate can be applied to apurification matrix such as a gel filtration medium.

Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAB) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. In addition, a chromatofocusing step canbe employed. Alternatively, a hydrophobic interaction chromatographystep can be employed. Suitable matrices can be phenyl or octyl moietiesbound to resins. In addition, affinity chromatography with a matrixwhich selectively binds the antibodies, variants or fragments can beemployed. In one embodiment a proteinA affinity chromatography column isused. Other examples of such resins employed are lectin columns, dyecolumns, and metal-chelating columns. Finally, one or morereversed-phase high performance liquid chromatography (RP-HPLC) stepsemploying apolar RP-HPLC media, (e.g., silica gel or polymer resinhaving pendant methyl, octyl, octyldecyl or other aliphatic groups) canbe employed to further purify the polypeptides. Some or all of theforegoing purification steps, in various combinations, are well knownand can be employed to provide an isolated and purified recombinantantibodies, variants or fragments.

Transformed yeast host cells are preferably employed to expressantibodies, variants or fragments as a secreted polypeptide in order tosimplify purification. Secreted recombinant antibodies, variants orfragments from a yeast host cell fermentation can be purified by methodswhich are well known to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the meaning commonly understood by a person who is skilled in theart in the field of the present invention.

Throughout the specification, unless the context demands otherwise, theterms ‘comprise’ or ‘include’, or variations such as ‘comprises’ or‘comprising’, ‘includes’ or ‘including’ will be understood to imply theinclusion of a stated integer or group of integers, but not theexclusion of any other integer or group of integers.

The invention being generally described, will be more readily understoodby reference to the following examples which are included merely forpurposes of illustration of certain aspects and embodiments of thepresent invention, and are not intended to limit the invention.

EXAMPLE 1 Production of CDR-Grafted Humanized R24 Antibodies

The hybridoma cell line R24, created from mice immunised with humanmelanoma cells (see Dippold, W. G. et al., Proc. Natl. Acad. Sci. USA(1980) 77 (10):6114-6118) secretes an IgG3 antibody which is specificfor the ganglioside GD3. The ganglioside GD3 is a tumour associatedantigen overexpressed on most human melanomas.

The gene sequence for the R24 antibody has been submitted to the NCBIdatabase: the kappa chain variable domain accession number is GI:6573606and the gamma chain variable domain accession number is GI:6573607(Kaminski, M. J. et al. J. Biol. Chem. (1999) 274 (9):5597-5604).

On analysis, the R24 kappa (vk) chain variable domain belongs to mousesubgroup 5 which corresponds to human subgroup 1 for the purpose of CDRgrafting. The canonical class of each CDR was identified: CDR-L1 belongsto canonical class 2 (form B) (Al-Lazikani, B., Lesk, A. M., Chothia, C.J., Mol. Biol. (1997) 273:927-948), and both CDR-L2 and CDR-L3 belong tocanonical class 1. The non-redundant database from NCBI was searched forsuitable human frameworks into which to graft the murine CDRs. The vkdesign was based on a human vk sequence with accession number GI:2597947 (Juul, L., Houghs, L., Barington, T., Immunogenetics (1998) 48(1):40-46). This belongs to human subgroup 1 and germline VkLa.

The R24 heavy (vh) chain variable domain was identified as belonging tomouse miscellaneous subgroup 3. CDR-H1 belongs to canonical class 1, andCDR-H2 belongs to canonical class 3. The CDR-H3 does not fall into anycanonical class, but is likely to form a kinked base according to therules of Shirai (Shirai, H., Kidera, A., Nakamura, H., FEBS Letters(1999) 455:188-197). The non-redundant database from NCBI was searchedfor suitable human frameworks into which to graft the murine CDRs. Thevh design was based on a human vh sequence with accession number 01:106423 (Shroeder, H. W and Wang, J. Y., Proc. Natl. Acad. Sci. USA(1990) 87:6146-6150). This sequence belongs to human subgroup 3 andgermline vh3-30.

The structure of the vk and vh sequences of R24 are available in theProtein Data Bank with codes IBZ7 and lR24, respectively (Kaminski, M.J. et al., J. Biol. Chem. (1999) 274 (9):5597-5604). These structureswere used to guide the choice of back mutations likely to give a betterfit for the grafted CDRs.

The CDR-grafted vk design for LD47:

(SEQ ID NO: 17) DIQMTQSPSSLSASVGDRVTITCRASQDIGNFLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGKTLPYTFGG GTKVEIK.In this sequence leucine 48 is back mutated to isoleucine as isoleucineis a canonical residue for CDR-L2.

The CDR-grafted vk design for LD49 is a straight CDR-graft with no backmutations:

(SEQ ID NO: 18) DIQMTQSPSSLSASVGDRVTITCRASQDIGNFLNWYQQKPGKAPKLLLYYTSRLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGKTLPYTFGG GTKVEIK.

The CDR-grafted vh design for LD47:

(SEQ ID NO: 19) QVQLVESGGGVVQPGRSLRLSCAASGFTFSNFGMHWVRQAPGKGLEWVAYISSGGSSINYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGGTGTRSLYYFDYWGQGTTVTVSS.

In this sequence alanine 93 is back mutated to threonine as this residuecontacts several residues in CDR-H3.

The heavy chain variable region in LD49 is that of the original murineantibody, no CDR grafting was involved, i.e., the LD49 gamma chain is achimeric mouse-human antibody chain.

Other back mutations were made and tested, however these were not foundto have any beneficial effect to the antibodies' affinity or effectorfunctions.

The sequences for the human IgG1 and kappa constant regions wereobtained from the NCBI database accession numbers G1:49522738 andG1:62531193 respectively (Strausberg, R. L. et aI., Proc. Acad. Sci. USA(2002) 99 (26):16899-16903). The LD47 antibody was made up of thefollowing heavy (gamma) and light (kappa) variable domain chains:

Chimeric Gamma:

(SEQ ID NO: 20) DVQLVES GGGLVQPGGS RKLSCAASGF TFSNFGMHWV RQAPEKGLEWVAYISSGGSS INYADTVKGR FTISRDNPKN TLFLQMTSLR SEDTAIYYCTRGGTGTRSLYYFDYWGQGATLIVSS.

CDR grafted Kappa 1 back mutation:

(SEQ ID NO: 21) DIQMTQSPSSLSASVGDRVTITCRASQDIGNFLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGKTLPYTFGG GTKVEIK.

The LD49 antibody was made up of the following heavy (gamma) and light(kappa) variable domain chains:

CDR grafted gamma one back mutation:

(SEQ ID NO: 22) QVQLVESGGGVVQPGRSLRLSCAASGFTFSNFGMHWVRQAPGKGLEWVAYISSGGSSINYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGGTGTRSLYYFDYWGQGITVTVSS.

CDR grafted Kappa, no back mutations:

(SEQ ID NO: 23) DIQMTQSPSSLSASVGDRVTITCRASQDIGNFLNWYQQKPGKAPKLLLYYTSRLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGKTLPYTFGG GTKVEIK.

The Glutamine Synthetase Gene Expression System uses the GS gene as aselectable marker to facilitate the high level expression of recombinantproteins. This allows the rapid generation of high-yieldingstably-transfected cell lines expressing recombinant antibodies.Double-gene plasmid constructs have been engineered for the LD47 andLD49 sequences above. Chinese Hamster Ovary (CHO) cells were transfectedby electroporation, and the resultant antibody-expressing clones wereexpanded.

For each recombinant antibody, 2 litres of culture material wasobtained. Purification of antibody from this material was achieved byprotein A affinity chromatography. The recovered antibodies were thenbuffer exchanged into PBS, pH 7.0. The concentrations of all bufferexchanged antibodies were recalculated by ELISA and optical densityreadings at 280 nm and 260 nm (using Beer-Lambert's Law A=ε.c.1; ε=1.4mL.mg⁻¹.cm⁻¹ for antibodies) and ranged from 0.56-1.29 mg/ml for ELISAresults and 0.53-1.08 mg/mL for 280 nm results, Table 1. Although adiscrepancy exists between ELISA and 280 nm concentration values thedifference between each result is relative see Table 1.

TABLE 1 Concentrations Of Buffer Exchanged Antibodies As Measured ByELISA And Optical Density ELISA A280 concentration concentrationAntibody Name Gamma Chain Kappa Chain mg/ml mg/ml LD47 CDR grafted 1CDR-grafted 1 0.85 0.7 back mutation back mutation LD49 Chimeric gammaCDR-grafted 1.001 1.07 no back mutation

EXAMPLE 2 Immunofluorescence Assay

Initial studies looked at binding of the humanized antibodies to humanmelanoma SK-MEL28 cells which express GD3. Binding of antibodies totarget cells was assayed by direct immunofluorescence using aFITC-labeled secondary antibody. Samples were analysed by one colourcytofluorimetric analysis.

Both LD47 and LD49 showed a high binding affinity to SK-MEL 28 cells. Inorder to overcome problems in one of the effector function assays adifferent GD3 expressing cell line was obtained, the human melanoma cellline MeWo. Several binding assays were repeated using this cell line theresults are shown in FIG. 1.

Antibodies were tested in separate experiments that gave different MLFresponses of the reference R24 antibody. To enable comparison betweenthe different tested antibodies, 30% of maximum binding of R24 wascalculated for each antibody. A figure of 30% was chosen for comparisonas some of the antibodies tested (not shown here) bound at low levels inthe concentration range tested and did not produce a higher relativeMLF. Especially as the antibodies show homophilic binding and do notsaturate the ganglioside GD3 antigen, the comparison gives a numericalindication of the relative abilities of the tested antibodies to bindganglioside GD3. The results are shown in Table 2.

TABLE 2 Binding And Cytotoxicity Assays Binding (30% of % cytotoxicityat Antibody Name maximum) (ug/ml) 10 ug/ml LD47 12.4 59.18 LD49 30.857.8

An experiment using immunofluorescence was also performed to compare thebinding of the LD49 antibody of the invention to SK-MEL28 cells with thebinding of the murine R24 antibody and a chimeric antibody published inChapman et aI., in Cancer Immunol. Immunother. (1994) 39: 198-204. TheLD49 antibody performed significantly better than the chimeric antibodyas illustrated by FIG. 2.

BIACORE Assay

Analyses were performed using a BIACORE 1000 biosensor system equippedwith a carboxymethyldextran-coated CM5 chip according to Catimel(Catimel et aI., 25 Glycobiology (1998) 8 (9): 927-938). Ganglioside GD3(1 mg/ml in methanol) was diluted 1:4 with HEPES buffer and injectedover the biosensor chip in order to immobilize the ganglioside. Theantibodies were then diluted to the required concentrations with HEPESbuffer and injected over the immobilized GD3. Biosensor curves wereobtained as shown in FIG. 3 for LD41, LD49 and the murine R24 antibody.

LD47 and LD49 bind to the GD3 with similar affinity as the murine R24antibody and not only do they display rapid association to the GD3, theyalso display slow dissociation.

GD3 ELISA

An ELISA to measure antibody binding to ganglioside GD3 was performed toconfirm that the antibodies were indeed binding to the ganglioside GD3.Plates were coated with human disialoganglioside GD3 and antibodiesadded to the plate over a range of concentrations. The antibodiesappeared to display varying degrees of binding to GD3 as was the casewith the binding assay and comparable results were obtained on eachoccasion the ELISA was performed. FIG. 4 illustrates one set of resultsobtained. These results correlate with the results from the bindingassay.

EXAMPLE 3

The humanized antibodies were assessed to determine their ability tomediate CDC. The ability of the humanized antibodies to fix humancomplement was examined using MeWo cells. In the complement assay,antibody diluted in medium was added to the MeWo cells. Human serumcomplement was also added to final concentration of 5% and the cellsincubated for 2 hours at 37° C. Subsequently, dead cells stained withpropidium iodide were identified by flow cytometry. Antibodyconcentrations from 100 ug/ml to 0.1 ug/ml were used. The results areshown in FIG. 5. In order to compare the humanized antibodies the %cytotoxicity at 10 ug/ml was calculated and is shown in Table 2.

EXAMPLE 4

In order to determine the homophilic properties of the humanizedantibodies their ability to bind to mouse R24 was measured. An ELISAplate was coated with the murine R24 and antibody binding was measuredover a range of concentrations. FIG. 6 shows the results of thehomophilic binding ELISA.

The results illustrate that LD47 and LD49 exhibit good homophilicbinding. This was repeated on three occasions and the results werecomparable.

EXAMPLE 5

ADCC was measured by the ability of the effector lymphocytes andmonocytes to lyse target cells. Histopaque-separated PBMCs (obtainedfrom buffy coats) were incubated with CFSE (carboxyfluorosceinsuccinimidyl ester)-stained MeWo cells and antibody. Subsequently, thedead cells were stained with propidium iodide and two colourcytofluorimetric analysis was performed. Initially, in order todetermine the ideal concentration of antibody and effector to target(E:T) ratio LD49 was assayed at 0.05-100 ug/ml and 100:1, 50:1 and 25:1effector to target ratios. FIG. 7 shows the results of this experiment.Above 12.5 ug/ml the antibodies' ability to mediate ADCC appears todecrease.

In order to compare the ability of the antibodies to mediate ADCC anideal E:T ratio and antibody concentration has to be determined. Forthis, the lead antibodies LD47 and LD49 were tested for their ability tomediate ADCC over a range of effector: target cell (E:T) ratios andantibody concentrations (See FIG. 8). From this an ideal E:T ratio of25:1 was chosen for all subsequent experiments.

The effect of R24, LD47 and LD49 at a range of antibody concentrationswas then investigated (FIG. 9). LD47 and LD49 showed high levels of ADCCmediation that were similar to the R24 murine antibody. PBMC-only andthe IgG1 isotype control both gave negative cytotoxicity values and arenot shown on the graph. The % cytotoxicity results in this experimentwere low, possibly due to the variation between buffy coats. In thisexperiment, the ability of the antibodies to mediate ADCC did notdecrease above 12.5 ug/ml as seen in FIG. 7.

This experiment was repeated on three occasions. FIG. 10 shows theresults for both with and without PBMCs and the LD47 and LD49 repeatedlydemonstrated efficient ADCC, which was superior to the murine R24antibody. In each experiment an E:T ratio of 25:1 was used and theantibodies were at a concentration of 10 ug/ml.

EXAMPLE 6 Phase I Studies of Humanized R24Antibodies

The following studies are designed to determine the toxicity,pharmacokinetics, tumor targeting, and immunogenicity of humanized R24antibodies. One goal is to select a phase II dose and schedule.

To be eligible for this trial, patients will be required to meet theusual eligibility criteria.

Major Eligibility Criteria

1. Patients with stage III or IV melanoma not curable by surgery.

2. ECOG performance status≧2. Life expectancy>3 months.

3. Prior therapy for melanoma will be allowed but all prior melanomatherapy must have been completed at least 2 weeks prior to startinghumanized R24 and all toxicities of prior therapy must have resolved.

4. Patients must have adequate organ and marrow function.

Major Exclusion Criteria

1. Prior treatment with a monoclonal antibody.

2. Concurrent immunosuppressive therapy (including steroids) or patientswho are likely to require steroids or immunosuppressive therapy in thenear future.

3. Patients with brain metastases will be excluded because of concernabout humanized R24 causing serious hypertension.

4. Since hypertension may be a DLT, patients with poorly-controlledhypertension are excluded.

5. Because of concern about of GD3 in the developing CNS, children under18 years of age, pregnant women, and women breast-feeding are excluded.

Treatment Plan

A cycle of treatment will consist of 4 weekly IV infusions of humanizedR24. Up to two additional treatment cycles can be administered after arest period of 2-4 weeks if toxicity is acceptable and there is noprogression of disease.

Dose levels: Three patients will be accrued into the followingescalating dose levels: 1, 5, 25, 125 mg/m². These dose levels werechosen based on the experience with mouse R24. If DLT is observed in 1patient, the cohort will be expanded to 6 patients. If DLT is observedin 2 or more patients at a dose level, that dose level will beconsidered to be above the DLT. In this case, a cohort of 3 patientswill be accrued at a dose level intermediate between the toxic doselevel and the prior dose level. If that intermediate dose level provesto be tolerated (i.e. <1 DLT), a final cohort can be accrued in a higherintermediate dose. On the other hand, if the first intermediate dose isnot tolerable, then a final cohort can be accrued in to a lowerintermediate dose. Up to 6 patients may be accrued into the cohortthought to represent the MTD in order to confirm safety.

Serum for pharmacokinetics will be collected pre-treatment and 1 hr, 2hrs, 4 hrs, 2 days, 4 days, and 7 days after the first treatment of eachtreatment cycle. Serum R24 levels can be measured by ELISA using BEC2murine anti-idiotypic MAb. Serum can also be assayed for humananti-human R24 antibody responses (HAHA). For this ELISA, F(ab′)₂fragments of humanized R24 will be prepared and used to coat 96-wellplates (1 μg/well). Bound human IgG can be detected using an alkalinephosphatase-conjugated second antibody specific for human Fc.

Pre Day 0 Day 2 Day 4 Wk 2 Wk 3 Wk 4 Wk 5 Wk 6 Humanized R24 treatment XX X X Physical exam X_(a) X X X CBC, comp X_(a) X X X X LDH X_(a) X CT,chest/abd/pelvis X_(b) X_(e) EKG X_(b) Brain MRI X_(b) Urinecatecholamines X_(c) Pharmacokinetics X_(d) X X X Anti-humanized R24 AbX X ₁₁₁In-Imaging X X X _(a)Within 2 weeks of starting treatment._(b)Within 4 weeks of starting treatment. _(c)Collected after treatmentprior to being discharged home. _(d)Pretreatment, 30 min, 1 hr, 2 hrs,and 4 hrs after completing the infusion. _(e)CT scan can be done anytime during weeks 5 or 6.Tumor Targeting Studies

A critical parameter to measure is how much MAb reaches the tumor.Patients will receive 6-8 mCi of In-111 chelated by 5 mg of DOTA-hu-R24per infusion. DOTA(1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid) can beconjugated to hu-R24 by random coupling of one of the four availablecarboxylic acids of DOTA to a terminal amine on the antibody. Themanufacture of this conjugate can be performed under cGMP conditions andthe material analyzed for sterility, pyrogen content, immunoreactivity,number of DOTAs attached per antibody, chelate stability, animalbiodistribution and acute toxicity.

Three patients in each cohort of the phase I trial will be imaged.Planar and/or SPECT images can be obtained 2-4 hours, 2-3 days and 4-6days after the end of infusion of radiolabeled HuR24. Blood samples forPK analysis can be collected at predose, 30 min, 60 min, 2 h, 4 h, 24 h,approximately 48 to 72 hours (2-3 days) and 96-144 hours (4-6 days)after each infusion of radiolabeled antibody. Samples will be analyzedusing RIA for concentrations of HuR24. Serum clearance will also bemeasured measuring ¹¹¹In.

EXAMPLE 7 Phase II Studies of Humanized R24Antibodies

The following studies are designed to test huR24 alone and incombination with other therapeutic agents. A goal is to select a regimenworthy of further testing in a randomized trial design.

The eligibility and exclusion criteria for these phase II trials will betypical for phase II trials (and similar to the criteria indicated abovefor the phase I trial) although prior non-cytotoxic therapy (e.g. IL-2,signal transduction inhibitors) would be allowed.

One phase II trial would certainly explore treatment with MAb alone.However, experience with other MAbs (e.g. rituximab, cetuximab,trastuzimab, bevacizumab) indicates that humanized R24 may be moreeffective in combination with an active chemotherapy regimen. Therefore,parallel phase II trials can be done to combine humanized R24 witheither temozolomide (TMZ) or a more active combination chemotherapyregimen such as CVD.

Other attractive agents to combine with huR24 would include, but are notlimited to:

β glucan: This sugar has been shown to enhance complement-mediatedcytotoxicity. It would be of interest to see if β glucan enhances theantitumor effects of huR24.

Ipilimumab: This anti-CTLA4 MAb activates T cells and has anti-melanomaactivity on its own. The drawback is that the T cell activation isnon-specific. By co-administering huR24, it may be possible to focus theinduced immune reactivity on GD3+ cells.

Biostatistics

Each of these phase II trials can be designed to use a Simon minimax twostage design in which a 10% response rate is considered not promising, a30% response rate is considered promising, and the probabilities of atype I error and type II error are set at 0.10. In the first stage ofthis design, 16 patients will be accrued to each cohort. If at least 2patients achieve a response among these 16 patients, then an additional9 patients will be accrued to the second stage. If <4 responses areseen, the study will be terminated and declared negative. This designyields at least a 0.90 probability of a positive result if the trueresponse rate is at least 30% and yields a 0.90 probability of anegative result if the true response rate is 10%.

1. An antibody that binds to the GD3 ganglioside antigen, comprising aheavy chain variable region that has the sequence of SEQ ID NO:24, and alight chain variable region that has the sequence of SEQ ID NO:2.
 2. Theantibody of claim 1, wherein the antibody is radiolabeled.
 3. Acomposition comprising the antibody of claim
 1. 4. The composition ofclaim 3, further comprising a therapeutic or an anti-melanoma drug. 5.The composition of claim 4, wherein the therapeutic or anti-melanomadrug is selected from the group consisting of one or more cytotoxicdrugs, interleukins, drugs that activate the immune system orlymphocytes, antibodies that bind to melanoma, antibodies that bind totumor matrix, antibodies designed to inhibit vascular structures, anddrugs that block critical biochemical pathways in melanoma.